Thermal cycling applications are an integral part of contemporary molecular biology. For example, the polymerase chain reaction (PCR), which is used to amplify nucleic acids, uses a series of DNA melting, annealing, and polymerisation steps at different temperatures to greatly amplify the amount of DNA in a sample. Conventional PCR reactions proceed in a closed vessel, with amplification being confirmed by extracting a sample from the finished reaction and analysing the product by gel electrophoresis.
This conventional analysis technique requires that the user wait until the cycling has finished before being able to confirm that amplification is taking place; this can lead to delays in obtaining experimental data, for example when a cycling reaction must be repeated due to failure of amplification. For this reason, alternative methods of analysing PCR and other amplification products have been developed which may be used to measure amplification at an earlier stage of the reaction. One such technique involves the incorporation of fluorescently labelled nucleotides into the reaction; as the DNA is amplified, so the intensity of fluorescence will increase. Detecting this fluorescence during the reaction can give a real-time indication of the progress of amplification. Many other molecular biology techniques make use of optical measurements to determine the progress of a reaction; for example, optical absorbance of a particular wavelength.
Measurement of fluorescence or other optical properties during progress of a reaction presents particular problems for the design of instrumentation and consumables. Conventional PCR reaction vessels are in the form of individual vessels having uniformly tapered conical portions, or take a multi-well plate format. Such vessels can present a relatively large cross section to illuminating and emitted light, so reducing the intensity of light able to be received at a detector. Further, the conical portions of such vessels enclose a relatively high volume of reaction mix, which therefore has a high thermal lag, leading to longer cycle times. Reduction in the volume of reaction mix can reduce this difficulty, but will reduce the amount of fluorescence produced by the reaction, so requiring more sensitive detectors. It is also necessary to include complex optical components in the thermal cycler to gather light emitted from the reaction vessel, and to reduce the effects of misalignment of the vessel and the light detector.
The effect of thermal lag is also exacerbated by the thickness of the reaction vessel walls. Thin walled vessels are available, having walls down to around 0.5 mm thick, but limits on injection moulding technology tend to prevent conventional reaction vessels being produced having substantially thinner walls.
Reaction vessels may be produced in the form of capillary tubes, but these require careful handling and transport to prevent unwanted damage to the vessel.
Various types of reaction vessels with removable lids have been described. For example, U.S. Pat. No. 5,720,406 describes a removable cover with a handle. U.S. Pat. No. 5,616,301 describes a holder for reaction vessels. U.S. Pat. No. 6,153,426 describes a thermal cycling apparatus with an integrated cover for closing reaction vessels. U.S. Pat. No. 6,620,612 describes a cover to be urged against a reaction vessel. EP 1 974 818 describes a cover affixable to a reaction vessel by heat sealing. U.S. Pat. No. 5,005,721 describes a vial seal which includes openings for securing the seal to a number of vials when the seal is open. WO 2006/024879 describes a thin walled reaction vessel with a generally flattened profile.
Embodiments of the present invention are intended to provide an alternative reaction vessel particularly suited for use in thermal cycling reactions.